Abstract

Background: Undisturbed functioning of the blood–brain barrier (BBB) crucially depends on paracellular signaling between its associated cells; particularly endothelial cells, pericytes and astrocytes. Hypoxic and ischemic injuries are closely associated with disturbed BBB function and the contribution of perivascular cells to hypoxic/ischemic barrier regulation has gained increased attention. Regardless, detailed information on the basal hypoxic/ischemic responses of the barrier-associated cells is rare and the outcome of such cell-specific responses on BBB modulation is not well understood. This study investigated crucial parameters of hypoxic/ischemic adaptation in order to characterize individual perivascular cell responses to stress conditions. Methods: The brain microvascular endothelial cell line RBE4 (EC cell line) as well as primary rat brain endothelial cells (ECs), pericytes (PCs) and astrocytes (ACs) were exposed to 24 and 48 hours of oxygen deprivation at 1% and 0.2% O2. All primary cells were additionally subjected to combined oxygen and glucose deprivation mimicking ischemia. Central parameters of cellular adaptation and state, such as HIF-1α and HIF-1 target gene induction, actin cytoskeletal architecture, proliferation and cell viability, were compared between the cell types. Results: We show that endothelial cells exhibit greater responsiveness and sensitivity to oxygen deprivation than ACs and PCs. This higher sensitivity coincided with rapid and significant stabilization of HIF-1α and its downstream targets (VEGF, GLUT-1, MMP-9 and PHD2), early disruption of the actin cytoskeleton and metabolic impairment in conditions where the perivascular cells remain largely unaffected. Additional adaptation (suppression) of proliferation also likely contributes to astrocytic and pericytic tolerance during severe injury conditions. Moreover, unlike the perivascular cells, ECs were incapable of inducing autophagy (monitored via LC3-II and Beclin-1 expression) - a putative protective mechanism. Notably, both ACs and PCs were significantly more susceptible to glucose than oxygen deprivation with ACs proving to be most resistant overall. Conclusion: In summary this work highlights considerable differences in sensitivity to hypoxic/ischemic injury between microvascular endothelial cells and the perivascular cells. This can have marked impact on barrier stability. Such fundamental knowledge provides an important foundation to better understand the complex cellular interactions at the BBB both physiologically and in injury-related contexts in vivo.

Abstract

Background: Undisturbed functioning of the blood–brain barrier (BBB) crucially depends on paracellular signaling between its associated cells; particularly endothelial cells, pericytes and astrocytes. Hypoxic and ischemic injuries are closely associated with disturbed BBB function and the contribution of perivascular cells to hypoxic/ischemic barrier regulation has gained increased attention. Regardless, detailed information on the basal hypoxic/ischemic responses of the barrier-associated cells is rare and the outcome of such cell-specific responses on BBB modulation is not well understood. This study investigated crucial parameters of hypoxic/ischemic adaptation in order to characterize individual perivascular cell responses to stress conditions. Methods: The brain microvascular endothelial cell line RBE4 (EC cell line) as well as primary rat brain endothelial cells (ECs), pericytes (PCs) and astrocytes (ACs) were exposed to 24 and 48 hours of oxygen deprivation at 1% and 0.2% O2. All primary cells were additionally subjected to combined oxygen and glucose deprivation mimicking ischemia. Central parameters of cellular adaptation and state, such as HIF-1α and HIF-1 target gene induction, actin cytoskeletal architecture, proliferation and cell viability, were compared between the cell types. Results: We show that endothelial cells exhibit greater responsiveness and sensitivity to oxygen deprivation than ACs and PCs. This higher sensitivity coincided with rapid and significant stabilization of HIF-1α and its downstream targets (VEGF, GLUT-1, MMP-9 and PHD2), early disruption of the actin cytoskeleton and metabolic impairment in conditions where the perivascular cells remain largely unaffected. Additional adaptation (suppression) of proliferation also likely contributes to astrocytic and pericytic tolerance during severe injury conditions. Moreover, unlike the perivascular cells, ECs were incapable of inducing autophagy (monitored via LC3-II and Beclin-1 expression) - a putative protective mechanism. Notably, both ACs and PCs were significantly more susceptible to glucose than oxygen deprivation with ACs proving to be most resistant overall. Conclusion: In summary this work highlights considerable differences in sensitivity to hypoxic/ischemic injury between microvascular endothelial cells and the perivascular cells. This can have marked impact on barrier stability. Such fundamental knowledge provides an important foundation to better understand the complex cellular interactions at the BBB both physiologically and in injury-related contexts in vivo.

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